The present invention relates to a novel alkaloid and novel bioactive alkaloid fractions derivable from Ribes preferably selected among Ribes Rubrum and Ribes nigrum; methods of manufacturing such bioactive Ribes alkaloid fractions and their use for the inhibition of IKK-β, PDE4 and/or PDE5 and in addition to their promoting effect on mitochondrial biogenesis and function; their therapeutic or non-therapeutic applications as nutritive or medicinal products in the management of conditions associated with impaired mitochondrial function or IKK-β, PDE4 and/or PDE5 activity, such as inflammation, neurodegeneration, dyslipidemia, type 2 diabetes mellitus, impaired wound healing, sarcopenia and other conditions associated with muscle dysfunction or tiredness and fatigue, or where optimization of muscular or cognitive function is desired; extracts, juices or concentrates of Ribes comprising such alkaloids; compostions comprising such alkaloids, including pharmaceutical compositions, nutritive products such as functional foods and nutraceutical compositions, cosmetic compositions and medical devices.
The global community is facing a serious challenge with the growing burden of lifestyle-related diseases like type 2 diabetes mellitus (DM2) and atherosclerotic cardiovascular disease (CVD). Binding together DM2 and CVD is the metabolic syndrome. Metabolic syndrome is characterized by a cluster of risk factors including atherogenic dyslipidemia, abdominal obesity, raised blood pressure, insulin resistance±glucose intolerance, a proinflammatory state and a prothrombotic state [Scott 2004, Circulation; 109:433-438]. The American Heart Association defines the metabolic syndrome as the combination of dyslipidemia, abdominal obesity, hypertension, and insulin resistance, a constellation of disorders that bestow a cardiovascular risk far greater than any of its individual components [Grundy 2004, Circulation; 109:433-438]. CVDs are the number one cause of death globally: more people die annually from CVDs than from any other cause. An estimated 17.3 million people died from CVDs in 2008, representing 30% of all global deaths. Of these deaths, an estimated 7.3 million were due to coronary heart disease and 6.2 million were due to stroke. By 2030, almost 23.6 million people will die from CVDs, mainly from heart disease and stroke. These are projected to remain the single leading causes of death [WHO Fact sheet No 317, September 2011]. CVD is associated with dyslipidemia leading to arterial plaque formation forming the basis for coronary heart disease and stroke. Of the 346 million people worldwide with diabetes, 90% have DM2. In 2004, an estimated 3.4 million people died from consequences of high blood sugar. WHO projects that diabetes deaths will double between 2005 and 2030 [WHO Fact sheet No 312, August 2011]. DM2 is characterized by insulin resistance and increased blood sugar levels and is highly associated with CVD [Moller 2001, Nature; 414, 821-27]. More and more attention is now being paid to combined atherogenic dyslipidemia which is typically presented in patients with DM2 and metabolic syndrome.
A fundamental treatment of CVD as well as DM2 is the reduction of the blood cholesterol, especially the LDL. Treatments for dyslipidemia includes statins, fibrates, inhibition of cholesterol absortion, inhibition of liver cholesterol synthesis, increased excretion of cholesterol and inhibition of free fatty acid release [Bhatnagar 2008, BMJ; 337:503-8]. Such existing treatments are associated with a number of adverse effects including muscle pain and damage which may be life threatening (rhambodmyolysis), as well as associated with liver damage and gastrointestinal side effects. Consequently, there is a strong need for effective therapeutic principles suitable for application in the broader population.
The wound healing process is complex and dynamic, restoring cellular structures and tissue layers. Acute wounds are either traumatic or surgical and move through the healing process at a predictable rate from insult to closure. Non-healing, or chronic wounds, are complex wounds that do not progress through the usual phases of healing. In non-healing wounds, changes occur within the molecular environment of a chronic wound that are not conducive to healing, such as high levels of inflammatory cytokines, and low levels of growth factors. These changes terminate the healing process and increase the potential for bacterial infections. Addressing the issues that might be responsible for the physiological wound changes may restart healing and diminish the risk of further complications, and a faster wound healing diminshes the time of exposure to bacteria and subsequent infections.
Treatment of various types of wounds represents a huge burden on health care systems and patients worldwide and an immense therapeutic challenge due to lack of effective treatment of chronic wounds.
71.5 million surgical procedures were performed in the United States alone in 2000. Unhealed acute wounds are open to infectious agents and infection occurs in approximately 10% of surgical wounds making a faster wound healing generally advantageous. Furthermore, many surgical patients are obese or have chronic diseases that cause an impaired wound healing, creating a great need for improved wound healing. An additional burden of acute wound healing is the challenge of scarring that may have long lasting functional, cosmetic as well as psychological consequences for the patient. A scar represents the sum of the injury, the reparative process and subsequent interventions to improve the scarring process. Both normal and hypertrophic scars remain difficult to treat and impossible to prevent and there is a great need for therapeutic principles that advance wound healing without problematic scarring.
Chronic non-healing wounds represent a silent epidemic that affects a large fraction of the world population and poses a major threat to the public health. In the United States alone, chronic wounds affect 6.5 million patients. In the Scandinavian countries, the associated costs account for 2-4% of the total health care expenses. The major chronic wound types are diabetic ulcers, pressure ulcers and venous ulcers.
It is estimated that there are over 7.4 million pressure ulcers in the world where estimation was possible i.e. excluding the vast number of developing countries. During the first two weeks of admission, hospital acquired pressure ulcers occur in approximately 9% of hospitalized patients and nearly 60,000 deaths occur annually in the United States from hospital-acquired pressure ulcers. Pressure ulcers can be a major source of infection and lead to complications such as septicemia, osteomyelitis and, even death. The healing of pressure ulcers take a long time and are costly and time-consuming to treat.
It is estimated that up to 25% of all diabetics will develop a diabetic foot ulcer and it is estimated that 12% of individuals with a foot ulcer will require amputation. About 71,000 non-traumatic lower-limb amputations were performed in the United States in people with diabetes in 2004. Treatment of diabetic foot ulcers is often a specialist task and represents a huge unsolved challenge in modern wound care.
Venous ulcers account for 70%-90% of ulcers found on the lower leg. Venous leg ulcerations present a common and recurring problem in older people creating discomfort and distress for the patient and a great cost to the health care services. In individuals 65 years and older, venous leg ulcers affect approximately 1.69% of the population. Up to one-third of treated patients experience four or more episodes of recurrence. The mainstay of treatment includes local wound care and continuous compression therapy by bandaging by trained personnel or by graduated compression stockings. However, compression therapy is contraindicated in those with occlusive arterial disease, it requires a trained staff to apply the compression bandages and patient compliance with compression stockings is often poor.
Many topical agents are available that are meant to improve the wound healing environment. Wound debridement removes devitalized tissue and accumulated debris and includes irrigation, excisional debridement, enzymatic debridement and biological debridement with maggot therapy. Topical therapy includes hyperbaric oxygen therapy, negative pressure wound therapy, application of growth factors such as platelet-derived growth factor, epidermal growth factor and granulocyte-macrophage colony stimulating factor, topical preparations with antiseptics and antimicrobials, including iodine, chlorhexidine, silver and antibiotics. Various wound dressings are used to manage the moisture level in and around the wound, including gauze bandages, fine mesh gauze impregnated with petroleum, paraffin wax, or other ointments, films, foams, alginates, hydrocolloids, hydrogels and hydroactive dressings. Due to the very nature of topical treatments, these address only the superficial aspects of wound healing and have proved insufficient for effective treatment. No effective oral therapy that improves wound healing is available. Therefore, there is a huge need for effective oral treatment that promotes wound healing from within.
To promote wound healing from within, various aspects of the wound healing process may be addressed. Healing is traditionally explained in terms of 4 overlapping phases: hemostasis, inflammation, proliferation, and maturation. During hemostasis, platelets play a crucial role in clot formation and the initial inflammatory aspect of tissue healing by secreting inflammatory cytokines and chemokines which subsequently attract leukocytes and macrophages to the site of injury. These cells débride injured tissue and secrete proteases, cytokines and growth factors that propagate various aspects of healing. During the proliferative phase, epithelialization, fibroplasia, and angiogenesis occur, forming granulation tissue, which includes inflammatory cells, fibroblasts, and neovasculature in a matrix of fibronectin, collagen, glycosaminoglycans, and proteoglycans. Finally, during the maturation phase, collagen forms tight cross-links to other collagen and with protein molecules, increasing the tensile strength of the scar. The entire wound healing process is highly complex and the cellular events that lead from open wound to scar formation overlap. A rich blood supply is vital to sustain newly formed tissue and angiogenesis is a key aspect of wound healing. It involves the release of numerous angiogenic molecules among which vascular endothelial growth factor (VEGF) secreted by macrophages and epidermal cells is critical for angiogenesis.
In relation to wound healing, phosphodiesterase 4 (PDE4) inhibition is a highly promising therapeutic strategy. cAMP is a second messenger involved in the cytokine production of inflammatory cells, in angiogenesis, and in the functional properties keratinocytes which are all relevant in the process of wound healing. The intracellular levels of cAMP are determined by the activities of adenylate cyclase which synthesize cAMP from ATP and PDE4, which hydrolyzes cAMP to AMP. PDE4 is expressed in a variety of cells including inflammatory cells, smooth muscle cells, fibroblasts, endothelial cells and keratinocytes [Bäumer 2007, Inflamm Allergy Drug Targets, March; 6(1):17-26] which are all present in the skin.
The effects of cAMP are transduced by two ubiquitously expressed intracellular cAMP receptors, protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC) [Whittmann 2013, P. Dermatol Ther, April 27; 3(1):1-15]. The cAMP/PKA signaling pathway has been demonstrated to promote endothelial cell sprouting and tube formation [Aslam 2013, Acta Physiologica; 207(694):O10] and cAMP acts as a second messenger in the release of VEGF, mediated by prostaglandin E2 (PGE2) through the cAMP/PKA signaling pathway [Ikari 2013, Am J Respir Cell Mol Biol, October; 49(4):571-81]. Activation of Epac through the cAMP/Epac signaling pathway has been demonstrated to attenuate thrombin-induced hyperpermeability in endothelial cells [Aslam 2013, Acta Physiologica 2013; 207(694):O10]. Endothelial progenitor cells (EPC) are centrally involved in angiogenesis in regenerating vasculature and the recruitment of these cells is in part mediated by a hypoxic gradient in the wound stimulating epidermal cells to enhanced expression of pro-angiogenic factors like stromal cell-derived factor-1α (SDF-1α) and VEGF [Ceradini 2004, Nat Med, August; 10(8):858-64], [Tepper 2005, Blood, February 1; 105(3):1068-77] which, subsequently, mobilize EPC from the bone marrow to the ischemic sites. Under hypoxic conditions such as in wounds, EPC are stimulated to form organized cell clusters, which then form cord-like vascular structures that undergo canalization and connect to existing vessels.
In chronic wounds, the process of angiogenesis is impaired, resulting in defective granulation tissue formation, which eventually causes failure of the wound healing to progress through the proliferation phase. For example, diabetic wounds are characterized by impaired wound healing associated with of decreased angiogenesis and VEGF expression in the wound [Bitto 2013, Clin Sci, December; 125(12):575-85], [Gu 2013, Diabetes Res Clin Pract; October; 102(1):53-9], [Asai 2006, J Invest Dermatol, May; 126(5): 1159-67] and it has been demonstrated that topical VEGF induces a significantly accelerated repair in experimental wounds in diabetic mice, and exogenous application of VEGF can increase early angiogenesis and tensile strength in the ischemic wounds in rats [Sinno 2013, Plast Surg Int; 2013:1-7]. Phosphodiesterase-4 inhibition augments human lung fibroblast VEGF production induced by prostaglandin E2 [Ikari 2013, Am J Respir Cell Mol Biol, October; 49(4):571-81] and topical administration of Sodium N-6,20-O-dibutyryl adenosine-30,50-cyclic phosphate (DBcAMP), a stabilized analog of cAMP in diabetic wounds enhances wound healing significantly [Asai 2006, J Invest Dermatol, May; 126(5): 1159-67]. It is therefore highly likely that inhibition of PDE4 may increase local VEGF-secretion and promote wound healing, in particular impaired wound healing.
Another important factor in the granulation phase is stromal cell-derived factor (SDF)-1α. SDF-1α plays a critical and multifaceted role in the wound-healing process in both normal and diabetic environments. It is a chemotactic factor regulating the migration of EPCs and angiogenesis. Hence upregulation of SDF-1α enhances wound healing [Nakamura 2013, Biomaterials, December; 34(37):9393-400] and decreased levels of SDF-1α impair healing by decreasing cellular migration and angiogenesis. Diabetic wounds are deficient in SDF-1α and increasing the level of SDF-1α increases diabetic wound healing [Bitto 2013, Clin Sci, December; 125(12):575-85], [Bermudez 2011, J Vasc Surg; 53:774-84]. Elevation of cAMP by local administration of DBcAMP in diabetic wounds has been demonstrated to increase the transcription and production of SDF-1α by macrophages and mesenchymal cells and significantly accelerate the wound healing [Asai 2006, J Invest Dermatol, May; 126(5):1159-67].
The proliferation of epidermal basal cells is another key aspect of wound healing. cAMP has long been regarded as a second messenger and a regulator of human keratinocyte proliferation. cAMP signaling regulates keratinocyte proliferation by modulating mitogen-activated protein kinase (MAPK) activity. DBcAMP has been demonstrated to promote the production of transforming growth factor-β by keratinocytes and fibroblasts, as well as the proliferation and migration of keratinocytes [Zhou 2000, Br J Dermatol, September; 143(3):506-12], [Onuma 2001, Arch Dermatol Res, March; 293(3):133-8], [Iwasaki 1994, J Invest Dermatol, June; 102(6):891-7] and accelerate healing and re-epithelialization of full-thickness wounds [Balakrishnan 2006, Biomaterials, March; 27(8):1355-61]. Similarly, elevation of cAMP by PDE4 inhibition may therefore enhance epithelialization in the process of wound healing.
PDE4 is expressed in cells such as endothelial cells, keratinocytes and fibroblasts [Bäumer 2007, Inflamm Allergy Drug Targets, March; 6(1):17-26] which are all present in the wound bed during wound healing. Topical application of a PDE4-inhibitor has been demonstrated to exert anti-inflammatory effects with reduced expression of cytokines and adhesion molecules [Ishii 2013, J Pharmacol Exp Ther, July; 346(1):105-12]. Topical administration of DBcAMP, another way to increase local cAMP, has been demonstrated to significantly reduce the inflammatory oedema in the arachidonic acid induced ear oedema model in mice [Rundfeldt 2012, Arch Dermatol Res, February 3(304):313-317]. The role of antiinflammatory effects elicited by PDE4 inhibition in supporting the wound healing may be most pronounced in chronic wounds where chronic inflammation is an important facet of the non-healing state of the wound and decreased inflammation is associated with increased wound healing [Eming 2007, J Invest Dermatol, March; 127(3):514-25]. Hence, modulation of pro-inflammatory mediators by PDE4 inhibition may add to the wound healing effects exerted by PDE4 inhibition through propagation of angiogenesis and enhanced epithelialization.
In conclusion, cAMP signaling is involved in the regulation of several functions of importance to wound healing including angiogenesis, inflammation and epithelialization. Elevation of cAMP through inhibition of PDE4 therefore is a highly relevant therapeutic strategy for enhancement of acute and chronic wound healing through modulation of pro-inflammatory mediators, propagation of angiogenesis and enhanced epithelialization.
In relation to enhanced wound healing, phosphodiesterase 5 (PDE5) inhibition is another highly promising therapeutic strategy. PDE5 is a phosphodiesterase capable of degrading cGMP to 5′-GMP thereby inhibiting the activity of cGMP. PDE5 inhibition prevents the degradation of cGMP, thereby enhancing and/or prolonging its effects. cGMP is a second messenger which may be synthesized as a result of nitric oxide (NO) activation of soluble guanylyl cyclase. It is involved in various physiological processes through the activation of protein kinase G (PKG). Conversion of cGMP to 5′-GMP by PDE5 effectively inhibits NO/cGMP signaling whereas PDE5 inhibition restores NO/cGMP signaling. NO is a small radical, formed from the amino acid L-arginine by three distinct isoforms of nitric oxide synthase. The inducible isoform (iNOS) is synthesized in the early phase of wound healing by inflammatory cells, mainly macrophages. However, many cells participate in NO synthesis during the proliferative phase after wounding. Beneficial effects of NO have been repetitiously demonstrated in wound healing, and may act through several mechanisms also including vasodilation, scavenging of oxidative stress components, improvement of angiogenesis and promotion of endothelial cell proliferation [Farsaei 2012, J Pharm Pharmaceut Sci; 15(4):483-498]. NO serves as an important mediator that regulates gene expression and proliferation in keratinocytes, regulation of fibroblast migration and collagen deposition in wounded tissue [Han et al 2012, Am J Pathol, April; 180(4):1465-73], [Frank et al 2002, Kidney International; 61: 882-888]. NO released through iNOS was shown to regulate collagen formation, cell proliferation and wound contraction in animal models of wound healing [Witte 2002, Metabolism, October; 51(10):1269-73]. Accordingly, protection and enhancement of the NO-cGMP-PKG signaling pathway by inhibition of PDE5-conversion of cGMP is indeed beneficial to wound healing as confirmed by the significantly improved wound-healing with the peroral PDE5 inhibitor Sildenafil in 15 different animal studies and 2 clinical human studies on hard-to-heal-wounds [Farsaei 2012, J Pharm Pharmaceut Sci; 15(4): 483-498]. Furthermore, PDE5 inhibition as a strategy for promoting angiogenesis has been demonstrated in relation to the PDE5 inhibitor Vardenafil, which upregulates protein expression of VEGF and enhance mobilization of EPC in peripheral blood and bone marrow, contributing to neovascularization in a model of unilateral hindlimb ischemia in mice [Sahara 2010, Arterioscler Thromb Vasc Biol, July; 30(7):1315-24]). This finding is supported by the in vitro and in vivo findings that endothelial progenitor cells express PDE5; that the PDE5 inhibitor tadalafil induces a significant increase in EPC number mediated by increased CXCR4 expression, and that prolonged therapy with PDE5 inhibitors in humans increases circulating EPC, supporting the notion of an involvement of cGMP second messenger system in both EPC release from the bone marrow and EPC-mediated peripheral re-endothelization. [Foresta et al 2005, Int J Impot Res, July-August; 17(4):377-80], [Foresta et al 2009, Clin Endocrinol, September; 71(3):412-6], [Foresta et al 2010, Curr Drug Deliv, October; 7(4):274-82]. In conclusion, PDE5 inhibition has been convincingly demonstrated to be a highly relevant therapeutic strategy in relation to enhancement of wound healing.
Another promising therapeutic target in wound healing is the mitochondria. EPCs are dysfunctional under diabetic conditions resulting in impaired peripheral circulation and delayed wound healing. It has been demonstrated that mitochondrial autophagy and mitochondrial impairment is induced in EPCs under high glucose condition, thus linking diabetic cardiovascular complications including impaired wound healing with dysfunctional mitochondria. Optimizing mitochondrial function could therefore also improve diabetic wound healing [Kim 2014, Biol Pharm Bull; 37(7):1248-52].
In relation to inflammatory disorders and conditions, PDE4 and IkappaB kinase β (IKK-β) inhibition are highly promising therapeutic strategies. PDE4 is the predominant cAMP degrading enzyme in a variety of inflammatory cells including eosinophils, neutrophils, macrophages, T cells and monocytes, and may increase the production of pro-inflammatory mediators such as TNF-α, IL-17, IL-22, and IFN-γ, and decrease anti-inflammatory mediators such as IL-10. Inhibition of PDE4 results in an elevation of cAMP in these cells, which in turn down-regulates the inflammatory response. The antiinflammatory effects of PDE4 inhibitors have been well documented both in vitro and in vivo and is mediated partly through PKA but is also associated with Epac, which appears to play a key role in suppressing unwanted inflammation [Parnell 2012, Br J Pharmacol; 166(2):434-46]. The PDE4 inhibitor Apremilast has profound anti-inflammatory properties in animal models of inflammatory disease, as well as human chronic inflammatory diseases such as psoriasis and psoriatic arthritis. It reduces complex inflammatory processes and interferes with the production of leukotriene B4, inducible nitric oxide synthase, matrix metalloproteinase and blocks the synthesis of several pro-inflammatory cytokines and chemokines, such as tumor necrosis factor alpha, interleukin 23, CXCL9, and CXCL10 in multiple cell types [Schett 2010, Ther Adv Musculoskelet Dis, October; 2(5):271-8], supporting the high relevance of PDE4 inhibition in various chronic inflammatory conditions of the skin, joints, lungs and intestines such as arthritis, psoriasis, chronic obstructive lung disease and inflammatory bowel diseases. Of further relevance to targeting PDE4, PDE4 deficiency suppresses macrophage infiltration in white adipose tissue and reduces adiposity, suggesting that PDE4 inhibitors could have utility in treatment of obesity and for suppression of obesity-induced inflammation in white adipose tissue [Ren 2009, Endocrinology; 150:3076-3082]. Inhibitors of cAMP-specific PDE4 has been shown to increase apolipoprotein A-I (apoA-I)-mediated cholesterol efflux up to 80 and 140% in human THP-1 and mouse J774.A1 macrophages, respectively, concomitant with an elevation of cAMP levels and may provide a novel strategy for the treatment of CVD by mobilizing cholesterol from atherosclerotic lesions [Lin 2002, Biochem Biophys Res Commun, January 18; 290(2):663-9]. PDE4 regulates cAMP pools that affect the activation/phosphorylation state of AMPK and PDE4 inhibition has been shown to activate AMPK [Omar 2009, Cell Signal, May; 21(5):760-6] [Park 2012, Cell, February 3; 148(3):421-33]. AMPK is a pivotal serine/threonine kinase participating in the regulation of glucose, lipid as well as protein metabolism and maintenance of energy homeostasis. Recent studies demonstrated that AMPK can also inhibit NF-κB, suppress the expression of inflammatory genes and attenuate inflammatory injury [Yao 2012, Sheng Li Xue Bao, June 25; 64(3):341-5]. In the liver, activation of AMPK results in enhanced fatty acid oxidation as well as decreased glucose production. The AMPK system may be partly responsible for the health benefits of exercise and is the target for the antidiabetic drug metformin. It is a key player in the development of new treatments for obesity, DM2, and the metabolic syndrome [Towler 2007, Circ Res, February 16; 100(3):328-41]. Thus, inhibition of PDE4 represents a promising therapeutic strategy in improving inflammatory conditions as well as metabolic conditions.
IKK-β is part of the upstream NF-κB signal transduction cascade of inflammation. IKK-β phosphorylates the inhibitory IκB protein resulting in dissociation of IκB from NF-κB. NF-κB is now free to migrate into the nucleus and activate the transcription of a cascade of proinflammatory cytokines [Häcker 2006, Sci. STKE; 357: 13]. Low-grade inflammation in different tissues is involved in metabolic disorders such as DM2 and CVD. In obesity, free fatty acid overload, endoplasmatic reticulum-overload and excessive glucose levels along with inflammatory macrophage infiltration in visceral fat resulting in chronic inflammation, activates IKK-β, leading to a viscious circle of continuous inflammation, induction of insulin resistance and enhanced VLDL-tricglyceride and lipoprotein production. The outcome on a macrophysiological level is hyperglycemia and hypertriglycerdemia [Meshkani 2009, Clin Biochem; 42 (13-14):1331-46], [Tsai 2009, Am J Physiol Gastrointest Liver Physiol; 296(6):G1287-98], [vDiepen 2011, J Lipid Res; 52:942-950], [Solinas, 2010, J Lipid Res; 24:2596-2611]. IKK-β has been found to serve as a critical molecular link between obesity, metabolic inflammation, and disorders of glucose homeostasis. IKK-β is activated by almost all forms of metabolic stress that have been implicated in insulin resistance or islet dysfunction. Furthermore, IKK-β is critically involved in the promotion of diet-induced obesity, metabolic inflammation, insulin resistance, and beta-cell dysfunction. Hypertriglyceridemia is caused by accumulation of VLDL particles in the plasma as a consequence of changes in lipid metabolism that are associated with obesity. The accumulation of lipids in numerous tissues is accompanied by increased inflammatory processes such as macrophage infiltration and production of inflammatory mediators in white adipose tissue. In liver, fat accumulation increases the activity of the pro-inflammatory NF-κB and liver-specific activation of NF-κB induces metabolic disturbances [Cai 2005, Nat Med; 11:183-90], [Arkan, 2005, Nat Med; 11:191-98]. Proinflammatory cytokines can cause hypertriglyceridemia and, conversely, suppression of inflammation may reduce hypertriglyceridemia [Goldfine 2008, Clin Transl Sci; 1:36-43] suggesting a direct causal role for inflammatory pathways in the development of hypertriglyceridemia. Specific activation of inflammatory pathways exclusively within hepatocytes induces hypertriglyceridemia and the hepatocytic IKK-β pathway has been identified as a possible target to treat hypertriglyceridemia. [Janna 2011, J Lipid Res; 52:942-50]. Furthermore, it has been shown that IKK-β inhibition reverses insulin resistance [Minsheng 2001, Science, August 31; 293(5535):1673-7] and inhibition of the IKK-β pathway enhances degradation of hepatic apoB100, revealing important links between modulation of the inflammatory IKK-β mediated signaling cascade and hepatic synthesis and secretion of apoB100-containing lipoproteins [Tsai 2009, Am J Physiol Gastrointest Liver Physiol, June; 296(6):G1287-98]. Thus, inhibition of IKK-β represents a promising therapeutic strategy in improving inflammatory conditions as well as hypertriglyceridemia and metabolic conditions.
Mitochondria are organelles in eukaryotic cells with their own genome that consume oxygen and substrates to generate ATP necessary for energy demanding processes. In aerobic cells the majority of ATP is produced by oxidative phosphorylation. In the mitochondria, electrons that are donated from the Krebs cycle are passed through the four complexes (complex I-IV) comprising the electron transport chain, eventually reducing oxygen and producing water. The flux of electrons creates an electrochemical potential between the intermembrane space and the matrix of the mitochondria. This potential is utilized by the ATP synthase to phosphorylate ADP producing ATP (oxidative phosphorylation). Mitochondria also participate in a wide range of other cellular processes, including signal transduction, cell cycle regulation, thermogenesis, and apoptosis. They are highly dynamic organelles that are continuously remodeling through fission, fusion, autophagy and biogenesis. Mitochondrial biogenesis is the expansion of existing mitochondrial content, whether through growth of the mitochondrial network (increase in mitochondrial mass) or division of preexisting mitochondria (increases in mitochondrial number). Mitochondrial biogenesis is triggered when the energy demand exceeds respiratory capacity e.g. in response to exercise, stress, hypoxia, nutrient availability, hormones including insulin, reactive oxygen production and temperature.
Spare respiratory capacity is the difference between ATP produced by oxidative phosphorylation at basal and that at maximal activity. Under certain conditions a tissue can require a sudden burst of additional cellular energy in response to stress or increased workload. If the spare respiratory capacity of the cells is not sufficient to provide the required ATP, affected cells risk being driven into senescence or cell death. Exhaustion of the reserve respiratory capacity has been correlated with a variety of pathologies including heart diseases, neurodegenerative disorders and cell death in smooth muscle [Desler 2012, Journal of Aging Research; 2012:p 1-9].
Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) is widely recognized as a principal regulator of mitochondrial biogenesis and function and therefor represents a highly interesting therapeutic direct or indirect target in relation to modulationg mitochondrial function. PGC-1α coactivates transcription factors that regulate expression of nuclear genes that encode mitochondrial proteins and also of the nuclear gene that encodes mitochondrial transcription factor A (TFAM), which regulates mitochondrial DNA transcription. Thus, PGC-1α regulates the coordinated expression of mitochondrial proteins encoded in both nuclear and mitochondrial genes, activating an array of transcription factors including activation of Nuclear Respiratory Factors 1 and 2 (NRF-1 and NRF-2) which regulate transcription of proteins in the respiratory chain, activation of PPAR-α which regulates enzymes for fatty acid oxidation (β-oxidation), activation of mitochondrial transcription factor A which activates expression of the mitochondrial genome leading to mitochondrial biogenesis, and coactivation of myocyte-enhancing factor 2A (MEF2A) which leads to increased insulin sensitivity by translocation of the glucose transporter to membrane leading to an improved glucose uptake.
Activation of PGC-1 α has been linked to the NO/cGMP signaling pathway which therefore represents a highly relevant strategy for modulating mitochondrial function [Nisoli 2004, Proc Natl Acad Sci, November 23; 101(47):16507-12] through the inhibition of PDE5. Long-term exposure to low concentrations of NO induces mitochondrial biogenesis mediated by cGMP, and involves increased expression of PGC-1α, NRF-1 and mitochondrial transcription factor A. [Nisoli 2003, Science, February 7; 299(5608):896-9.]. NO/cGMP dependent mitochondrial biogenesis furthermore yields functionally active mitochondria, in terms of respiratory function and metabolic activity [Nisoli 2004, Proc Natl Acad Sci, November 23; 101(47):16507-12]. Therefore, inhibition of PDE5, which results in increased levels of cGMP is a very interesting target for stimulation of mitochondrial biogenesis and functionality. This is supported by the finding that cGMP-selective phosphodiesterase inhibitors stimulate mitochondrial biogenesis in kidney tissue [Whitaker 2013, J Pharmacol Exp Ther, December; 347(3): 626-34] and short term PDE5-inhibition with the PDE5 inhibitor Sildenafil has been shown to reduce muscle fatigue and increase skeletal muscle protein synthesis [Sheffield More et al 2013, Clin Transl Sci, December; 6(6):463-8].
Like PDE5 inhibition, inhibition of PDE4, has also been linked to activation of PGC-1α and to stimulation of mitochondrial biogenesis and increased endurance, though through different pathways. Hence, the PDE4 inhibitor Rolipram has been demonstrated to induce mitochondrial biogenesis and increase the expression of PGC-1α, as well as inducing a significantly greater distance on a treadmill before exhaustion in Rolipram treated mice than control mice [Park 2012, Cell, February 3; 148(3):421-33].
It is well established that endurance exercise training induce large increases in mitochondria and even a single bout of exercise induces a rapid increase in mitochondrial biogenesis that is mediated both by activation and by increased expression of PGC-1α [Hollzy 2011, Compr Physiol, April; 1(2):921-40], [Holloszy 2008, J Physiol Pharmacol, December; 59 Suppl 7:5-18][Bartlett 2012, J Appl Physiol, April; 112(7): 1135-43]. PGC-1α signaling controls mitochondrial biogenesis and angiogenesis in response to endurance exercise in skeletal muscle and PGC-1α has been shown to increases exercise performance [Tadaishi et al 2011, PLoS ONE, December, Vol 6, Issue 12:1-13] and to a large degree, the adaptive changes in skeletal muscles such as fiber type transformation, mitochondrial biogenesis, angiogenesis, improved insulin sensitivity and metabolic flexibility induced by endurance training is regulated by PGC-1α [Lira 2010, Am J Physiol Endocrinol Metab, August; 299(2):E145-61], [Calvo et al 2008, J Appl Physiol 2008 May; 104(5):1304-12]. Thus, increasing mitochondrial function and biogenesis is a highly relevant strategy for improving exercise and endurance performance in relation to sport.
Aging is an inevitable biological process characterized by the progressive deterioration of a variety of physiological functions, rendering the aging person increasingly frail and susceptible to diseases. The aging process is linked to increasingly dysfunctional mitochondria by a decrease in the rate of mitochondrial oxidative phosphorylation, increase in the capacity of mitochondria to produce ROS, and impairment of the mitochondrial oxidation of specific substrates. As a result, these age-induced alterations in mitochondrial function impair energy production as well as increase the production of toxic reactive oxygen intermediates [Marcovina 2013, Transl Res. February; 161(2):73-84]. The age-related decline of mitochondrial capacity for oxidative phosphorylation and accumulation of mitochondrial DNA mutations has been linked to the pathogenesis of a range of age-related pathological alterations including alopecia, osteoporosis, kyphosis, cardiomyopathy, anemia, gonadal atrophy and sarcopeniea [Desler 2012, Journal of Aging Research, 2012:1-9], and mitochondria dysfunction has been linked to most age-related diseases such as neurodegeneration, cardiovascular disease and diabetes.
Reductions in skeletal muscle function occur during the course of healthy aging as well as with bed rest or diverse diseases such as cancer, muscular dystrophy, and heart failure. Muscle fatigue as symptom of reduced muscle function is a common symptom during sport and exercise activities, but is also increasingly observed as a secondary outcome in many diseases and health conditions during performance of everyday activities. However, there are no accepted pharmacologic therapies to improve impaired skeletal muscle function. Thus, within aged or sedentary skeletal muscle, there is a significant loss in the number of fibres and demonstrable biochemical and morphological abnormalities. Several large-scale studies on skeletal muscle biopsies from humans of ages ranging from 17 to 91 years have shown a significant age-related decline in mitochondrial respiratory capacity. The substantial fall in mitochondrial respiratory capacity in ageing muscle may contribute to the reduced exercise capacity in elderly people and the associated increased risk of diseases associated with an increasingly sedentary life style. Also, mitochondrial changes may underlie not only a loss of muscle function with age, but also other common age-associated pathologies increasing the risk of disease such as ectopic lipid infiltration, systemic inflammation, and insulin resistance. [Desler 2012, Journal of Aging Research, 2012:1-9], [Scheibye-Knudsen et al. 2013, Aging, March, Vol. 5 No. 3:192-208], [Peterson et al 2012, Journal of Aging Research, p 1-20], [Boffoli et al 1994, Biochim Biophys Acta., April 12; 1226(1):73-82]. As previously mentioned, PGC-1α is a key regulator of mitochondrial biogenesis and function, and it has been shown that lifelong training preserves mitochondrial DNA and PGC-1α whereas lifelong sedentary behavior reduces such markers of mitochondrial content. Furthermore, it has been shown that despite the mitochondrial dysfunction observed with sedentary ageing, muscles from sedentary elderly individuals retain the capacity to activate the acute signaling pathways associated with regulating the early processes of mitochondrial biogenesis [Cobley 2012, Biogerontology. 13(6):621-631]. Hence, improvement of mitochondrial biogenesis and function for instance through activation of PGC-1α by inhibition of PDE4 and PDE5 in the elderly as well as during bed rest or diseases or conditions that impairs muscular function, is highly relevant.
The central nervous system is particularly prone to mitochondrial dysfunction and augmentation of mitochondrial function may play a pivotal role in a range of CNS-disorders. Exhausting the reserve respiratory capacity of a neuron can have fatal consequences. Resting neurons utilize approximately 6% of its maximal respiratory capacity, while firing neurons utilize up to 80%. Therefore, subtle aging-related decreases in spare respiratory capacity increase neuronal vulnerability towards bioenergetic exhaustion, predisposing the tissue for diseases. Hence, mitochondrial abnormalities occur in persons with various neurodegenerative diseases and distinct mitochondrial abnormalities are characteristic of particular disorders. This is the case for common age-related disorders such as Alzheimer's disease. Alzheimer's disease is a major problem in the global aging population with more than 25 million people affected by dementia, most suffering from Alzheimer's disease. In the United States alone, Alzheimer's disease affects approximately 5.4 million people and the number is projected to reach 12-16 million by the year 2050. In the United States in 2011, the cost of health care, long-term care, and hospice services for people aged 65 years and older with Alzheimer's disease and other dementias was expected to be $183 billion. Increasing evidence links Alzheimer's disease with mitochondrial dysfunction. Rodent models of the neurodegenerative Alzheimer's disease show that deficiency in mitochondrial respiration precedes the pathology of the disease. Alzheimer's disease is also accompanied by decreased expression and activity of enzymes involved in mitochondrial bioenergetics. Correspondingly, a decline of brain metabolism is detectable in Alzheimer's disease patients as early as a decade before diagnosis. Besides functional changes, extensive literature indicates mitochondrial structural dynamics are also altered in Alzheimer's disease patients. Other neurodegenerative diseases also linked to mitochondrial dysfunction are Parkinson's disease, ALS motor neuron degeneration and Huntington's disease [Lezi 2012, Adv Exp Med Biol; 942:269-286], [Desler 2012, Journal of Aging Research; 2012:1-9].
Ribes is a genus of about 150 species of flowering plants native throughout the temperate regions of the Northern Hemisphere. It is usually treated as the only genus in the family Grossulariaceae. The species Ribes rubrum and Ribes nigrum are widely cultivated due to their production of the edible redcurrants, blackcurrants, greencurrants and whitecurrants. A variety of subspecies and numerous cultivars are recognized. These berries have a widespread utility in the food and beverages industries, e.g. in the form of juice.
In 2002 Lu et al discovered the two nitrile alkaloids nigrumin-5-p-coumarate [systematic name (E)-(E)-2-cyano-4-(β-D-glucopyranosyloxy)but-2-en-1-yl 3-(4-hydroxyphenyl)acrylate] and nigrumin-5-ferulate [systematic name (E)-(E)-2-cyano-4-(β-D-glucopyranosyloxy)but-2-en-1-yl 3-(4-hydroxy-3-methoxyphenyl)acrylate] in the seeds of Ribes nigrum [Lu 2002, Phytochemistry; 59(4):465-8].
In 2007 Schwartz et al discovered the two nitrile alkaloids with the systematic names (E)-2-cyano-4-(β-D-glucopyranosyloxy)but-2-en-1-yl 4-hydroxy-3-methoxybenzoate and (E)-2-cyano-4-(β-D-glucopyranosyloxy)but-2-en-1-yl′4-hydroxybenzoate together with the indole alkaloids with the systematic names 1-β-D-glucopyranosyl-1H-indole-3-acetic acid and 1-β-D-glucopyranosyl-1H-indole-3-acetic acid methyl ester, which were all observed to contribute to the bitter taste of redcurrants [Schwarz 2007, J Agric Food Chem; 55:1405-1410].
None of the alkaloids found in the mentioned Ribes species have been attributed to any medicinal properties.